CN114014892A - BODIPY fluorescent probe for specifically recognizing superoxide anion and preparation method and application thereof - Google Patents
BODIPY fluorescent probe for specifically recognizing superoxide anion and preparation method and application thereof Download PDFInfo
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- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000000523 sample Substances 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
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- 238000001035 drying Methods 0.000 claims description 9
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
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- -1 superoxide anions Chemical class 0.000 abstract description 23
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
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- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention relates to a BODIPY fluorescent probe for specifically recognizing superoxide anions, and a preparation method and application thereof. In addition, the probe can realize detection of superoxide anion in an extremely wide pH range, and the fluorescence of the probe is not influenced by the pH of the environment, which is very beneficial to the practical application of the probe; the probe BOIPDY-T has excellent optical property, low pH sensitivity, high sensitivity and high selectivity, and can be easily applied to the field of chemical analysis and detection.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to a BODIPY fluorescent probe for specifically recognizing superoxide anions and a preparation method and application thereof.
Background
Among various Reactive Oxygen Species (ROS) in the living body, oxygen (O) passing through the respiratory chain of mitochondria2) The generated superoxide anion is an extremely important sterilization oxidant in organisms and plays a role in protecting human health. However, excessive superoxide anion production has been implicated in a range of pathological conditions such as alzheimer's disease, rheumatoid arthritis, heart disease, diabetes, and even cancer. In addition, the lifetime of superoxide anions generated in vivoVery short, and typically converted to other ROS over tens of microns of the production site. Although the change in superoxide anion concentration plays a very important role in vivo, its low concentration and short lifetime make detection of superoxide anions very difficult. Therefore, specific measurement and tracking of superoxide anions in complex biological systems is of great interest to elucidate the exact pathogenesis of the associated disease. So far, many chemical small molecule fluorescent probes for superoxide anions have been reported. For example, publication numbers CN107337681A, CNCN111100184A, etc.
However, these prior art techniques are not BODIPY-based probes. The BODIPY family member is used as a large fluorescent dye, and has been widely applied to functional fluorescent biological probe mother cores due to the excellent spectral and photophysical properties including excellent environmental stability, large molar extinction coefficient, high quantum yield and the like. Up to now, the fluorescent biological probe mother nucleus does not realize the application of the excellent performance of BODIPY to the specific recognition of superoxide anion, and thus needs to be improved.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to overcome the shortcomings in the prior art, and provides a BODIPY fluorescent probe capable of specifically recognizing a superoxide anion, and a preparation method and an application thereof.
The application provides a BODIPY fluorescent probe capable of specifically recognizing superoxide anion, and the molecular formula of the fluorescent probe is C33H30BF2N2O4P, the structural formula is as follows:
a preparation method of a BODIPY fluorescent probe for specifically recognizing superoxide anions comprises the following steps:
dissolving a mixture of T-Br, BODIPY-COOH and DBU in anhydrous tetrahydrofuran to prepare a mixed solution A, stirring at room temperature under the protection of argon, and performing reduced pressure spin drying on the solvent after the reaction is finished; further purifying by column chromatography, eluting with eluent to obtain probe BODIPY-T;
wherein the structural formula of T-Br is
Wherein the structural formula of BODIPY-COOH is shown as
Wherein DBU has a structural formula of
Further, the preparation method of the T-Br comprises the following steps:
s1, mixing and reacting raw materials: dissolving a mixture of T-OH, NBS and DMTU in dichloromethane to prepare a mixed solution B with the volume molar concentration of 0.1-1.0mol/L, wherein the dichloromethane also plays a role in diluting reactants, and stirring at room temperature to obtain a reaction solution;
s2, preparation of intermediate T-Br: washing the reaction solution with water for 3 times, drying dichloromethane with anhydrous sodium sulfate, and finally, carrying out decompression spin-drying on the solvent to obtain an intermediate T-Br;
wherein the structural formula of T-OH is
Wherein NBS has the structural formula
Wherein the structural formula of DMTU is
Further, the eluent is a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 2: 1.
Furthermore, the molar ratio of the T-Br, the BODIPY-COOH and the DBU is 9:5: 10.
Further, the molar ratio of T-OH, NBS and DMTU is 1:1.5: 0.45.
Further, the room temperature is 15-30 ℃, preferably 25 ℃.
Further, the volume molar concentration of the mixed solution A is 0.5-1.0 mol/L.
The application of the BODIPY fluorescent probe specifically recognizing the superoxide anion in a detection reagent for detecting and recognizing the superoxide anion in the environment or a biological sample, determination of the concentration of the superoxide anion in water or a marker.
Further, the application of the compound in preparing a detection reagent or a marker of superoxide anions in normal cells and living bodies.
Further, the detection method for detecting the superoxide anion by the BODIPY fluorescent probe of the superoxide anion comprises the following steps:
measuring the fluorescence intensity of superoxide anion at the wavelength of 530nm by using a fluorescence spectrophotometry with 480nm as an excitation wavelength; the detection limit of the sample was 0.062 micromolar.
By adopting the detection method: the fluorescence probe disclosed by the invention has weak fluorescence, can rapidly emit strong fluorescence after reacting with superoxide anions, and has a maximum emission wavelength of 530nm, the Stokes shift of the fluorescence probe disclosed by the invention is about 60nm, the maximum absorption wavelength of the fluorescence probe is near 470nm, and the maximum emission wavelength of the fluorescence probe after reacting with the superoxide anions is 530 nm.
To sum up, the application comprises the following beneficial technical effects:
1. esterification of BODIPY-COOH is a unique technique for designing fluorescent probes because the esterified derivative is red-shifted in absorbance and drastically reduced in fluorescence quantum yield, while the carboxylated derivative is blue-shifted in absorbance and highly fluorescent. In the invention, BODIPY-COOH is used as a mother nucleus, diphenyl phosphate is used as a superoxide anion recognition group to design and synthesize a novel superoxide anion fluorescent probe (BODIPY-T) which is used for detecting endogenous superoxide anions in living cells in an ultrasensitive and selective manner without being interfered by intracellular redox agents and pH values;
2. the fluorescent probe can also be used for the research of the determination of the concentration of superoxide anions in water and the imaging of the superoxide anions in cells;
3. the fluorescent probe has good selectivity on superoxide anion, the fluorescence intensity of the probe solution is weak in PBS (phosphate buffer solution) with the pH value equal to 7.4, 20 mu M potassium superoxide is added, the fluorescence intensity is gradually enhanced, and the fluorescence intensity is increased to about 12.9 times of the original fluorescence intensity after 5 minutes. Under the same condition, other possibly interfered amino acids and ions are respectively added, the fluorescence intensity of the probe is not obviously changed, and the probe has high selectivity on superoxide anions;
4. the fluorescent probe provided by the invention has high sensitivity on detection of superoxide anions. The fluorescence intensity of the probe solution increases with increasing superoxide anion concentration, and peaks at approximately 4-fold superoxide anion addition. In the interval of 0 to 2.4 times of superoxide anion, the fluorescence intensity of the probe solution has good linear relation with the concentration of the superoxide anion;
5. the probe can accurately detect superoxide anions in a very wide pH range, and the self-fluorescence is not influenced by pH;
6. the BODIPY derivative is used as a fluorescent parent nucleus of a probe, and the structure of the BODIPY derivative determines that the BODIPY derivative has excellent fluorescent property. The fluorescence of the probe formed by modification can be almost completely masked, and after the probe reacts with superoxide anion for several minutes, the fluorescence intensity is rapidly enhanced (the fluorescence enhancement multiple is greater than 12 times), and the quantum yield is very high. The detection limit is as low as 0.062 micromole, the sensitivity is high, and the selectivity and the anti-interference performance are good;
in conclusion, the fluorescent probe is a convenient and sensitive tool, is suitable for detecting superoxide anions in vitro and inside living cells, and has wide application prospects in the field of chemical analysis and detection.
Drawings
FIG. 1 is a schematic diagram of a process for preparing a fluorescent probe according to the present invention;
FIG. 2 is a mass spectrum of intermediate T-Br of the present invention;
FIG. 3 is a nuclear magnetic hydrogen spectrum of the fluorescent probe BODIPY-T of the present invention;
FIG. 4 is a nuclear magnetic carbon spectrum of the fluorescent probe BODIPY-T of the present invention;
FIG. 5 is a mass spectrum of the fluorescent probe BODIPY-T of the present invention;
FIG. 6 is an absorption spectrum and a fluorescence spectrum of a fluorescent probe (5. mu.M) of the present invention after adding superoxide anion (0-20. mu.M) at increasing concentrations to a PBS: DMSO 4:1 buffer solution (pH 7.4) and reacting for 5 minutes;
FIG. 7 is a diagram illustrating the reaction mechanism of the fluorescent probe of the present invention with superoxide anion;
FIG. 8 is a graph showing the change in fluorescence intensity of the fluorescent probe of the present invention (5. mu.M) after 5 minutes of reaction with increasing concentrations of superoxide anion (0-20. mu.M) in PBS: DMSO 4:1 buffer (pH 7.4). The inset is a linear plot of increasing concentrations of superoxide anion (0-12 μ M) added to fluorescent probes of the invention (10 μ M) in PBS: DMSO 4:1 buffer (pH 7.4);
FIG. 9 is a bar graph of various ion and molecular selectivities of the fluorescent probes of the present invention. Wherein, the color bars represent the fluorescence intensity after the following substances (20 μ M) and the probe (5 μ M) are reacted respectively: manganese chloride, sodium sulfate, silver nitrate, ferrous sulfate, ferric sulfate, cysteine, glutathione, sodium sulfide, sodium bicarbonate, sodium hydrosulfide, sodium sulfate, hydrazine hydrate, nitric acid, hypochlorous acid, hydrogen peroxide, potassium superoxide and water;
FIG. 10 is a graph showing the response of the fluorescent probe of the present invention to superoxide anion and the change in fluorescence under different pH environments;
FIG. 11 is a graph showing the MTT toxicity test of the fluorescent probe of the present invention as a function of the concentration gradient (final concentration) of the probe;
FIG. 12 shows the imaging results of the fluorescent probe (5. mu.M) of the present invention in RAW264.7 cells under different conditions;
fig. 13 is a two-dimensional code of the color artwork of fig. 6-12.
Detailed Description
The present application is described in further detail below with reference to figures 1-13.
The embodiment of the application discloses a preparation method of a BODIPY fluorescent probe for specifically recognizing superoxide anions.
The synthetic route of the fluorescent probe is shown in FIG. 1.
A mixture of T-OH (325mg,1.0mmol,1.0eq.) and NBS (265mg,1.5mmol,1.5eq.) was dissolved in 5mL of dichloromethane, DMTU (47mg,0.45mmol,0.45eq.) was added, the volume molar concentration of the mixed solution was 0.59mol/L, and the mixture was stirred at room temperature for 2 hours. After the reaction, the mixed solution was diluted with dichloromethane, then washed with water three times, followed by drying dichloromethane over anhydrous sodium sulfate, and finally spin-drying the solvent under reduced pressure to obtain 227mg of intermediate T-Br with a yield of 70%. The mass spectrum is shown in FIG. 2.
HRMS(ESI)calculated for C19H17BrO2P+,[M+H]+,387.0144,found,387.0142.
Intermediate T-Br (193mg,0.45mmol), BODIPY-COOH (75.0mg,0.25mmol) was dissolved in 5mL of anhydrous tetrahydrofuran, and DBU (76.0. mu.L, 0.5mmol) was added thereto, whereupon the volume molar concentration of the mixed solution was 0.24mol/L, followed by stirring under argon at room temperature overnight for an hour. After the reaction was complete, the tetrahydrofuran was removed by rotary drying under reduced pressure, the residue was dissolved in dichloromethane, washed twice with 1N aqueous hydrochloric acid, dried over sodium sulfate and evaporated to dryness. Further purification by column chromatography and purification of the residue on silica gel using petroleum ether/ethyl acetate (2/1v/v) as eluent gave probe BODIPY-T82 mg, 55% yield. The nuclear magnetic spectrum and the mass spectrum are shown in figures 3-5.
1HNMR(400MHz,CDCl3)δ＝7.92-7.89(m,2H),7.89-7.86(m,2H),7.58-7.53(m,2H),7.50-7.45(m,4H),7.33(d,J＝8.4Hz,2H),7.25(d,J＝8.4Hz,2H),6.00(s,2H),5.29(s,2H),2.51(s,6H),1.90(s,6H).13CNMR(101MHz,CDCl3)δ＝164.9,157.7,151.5,141.2,132.7,131.9,131.8,131.0,130.0,128.8,128.7,121.2,121.13,121.08,67.9,14.8,12.6.HRMS(ESI)calculated for C33H31BF2N2O4P+,[M+H]+,599.2077,found,599.2076.
The details of the detection mechanism of the fluorescent probe of the invention on superoxide anion are as follows: after the carboxyl group of BODIPY is esterified, the strong electron-withdrawing effect of the ester group causes the fluorescence to be masked. After the phosphate ester of BODIPY-T is oxidized by superoxide anion, intramolecular elimination reaction is caused, ester bond breakage is caused, carboxyl is exposed, and strong fluorescence is emitted. The absorption and fluorescence change of the probe in the response process are shown in FIG. 6, and the reaction mechanism of the fluorescent probe and superoxide anion is shown in FIG. 7.
Experimental analysis:
correlation between fluorescence intensity and superoxide anion concentration
Taking 5 mu M PBS (fluorescent probe) of DMSO 4:1 buffer solution (pH is 7.4), adding increasing concentration of superoxide anion (0-20 mu M), respectively having 0 mu M,2 mu M, 4 mu M, 6 mu M, 8 mu M, 10 mu M, 12 mu M, 14 mu M, 16 mu M, 18 mu M and 20 mu M, reacting for 5 minutes, the fluorescence spectrogram is shown in figure 8a, the linear relation graph of the fluorescence intensity and the concentration of the superoxide anion is shown in figure 8b, calculating to obtain the detection limit of the molecule which can reach 0.062 micromole and is suitable for micro-detection. Therefore, the fluorescent probe has good application prospect.
Second, fluorescent probe anti-interference ability detection
Taking a probe molecule solution (5 mu M) to prepare a solution to be detected in a PBS (PBS: DMSO 4: 1) buffer solution (pH is 7.4), adding various interferent molecules, manganese chloride, sodium sulfate, silver nitrate, ferrous sulfate, ferric sulfate, cysteine, glutathione, sodium sulfide, sodium bicarbonate, sodium hydrosulfide, sodium sulfate, hydrazine hydrate, nitric acid, hypochlorous acid, hydrogen peroxide, potassium superoxide and water respectively, and measuring after reacting for 5 minutes, wherein the result is shown in figure 9, the fluorescence of the solution is hardly changed obviously, and the fluorescence of the probe solution only added with superoxide anions is greatly enhanced (about 12 times), so that the fluorescent probe can realize special identification on the superoxide anions. The fluorescent probe has strong anti-interference capability on the detection of superoxide anions.
Thirdly, the response condition and the fluorescence change of the fluorescent probe to the superoxide anion in different pH environments
Probe molecule solutions (5 μ M) were added to phosphate buffers of different pH ( pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) to prepare solutions to be tested, and fluorescence intensities thereof were measured, and 20 μ M potassium superoxide was added thereto, and the results of the measurement after 5 minutes of reaction were shown in fig. 10. The fluorescence intensity of the probe molecule is kept stable in buffer solutions with different pH values, and the fluorescence is enhanced by about 12 times after the probe molecule is buffered with potassium superoxide in different pH values, and the fluorescence intensity is not influenced by the pH value. It can be seen that the fluorescent probe of the invention has good environmental pH interference resistance.
MTT toxicity test of fluorescent probe
RAW264.7 cells were plated in 4-well dishes (1.3X 10)4One/well), placed in a cell culture box, and completely attached to the wall. Then, the fresh culture solution was replaced, 200. mu.L of fluorescent probe dispersions of different concentrations, 100. mu.M, 80. mu.M, 60. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, and 0. mu.M, were added, and after 24 hours of culture, the wavelength was set to 570nm on a microplate reader, the absorbance (OD value) of the solution per well of a 96-well plate was measured, and the cell survival rate was calculated according to the following formula: the cell survival rate was (OD test group-OD blank)/(OD cell group-OD blank) x 100%. As can be seen in FIG. 11, the cell viability was greater than 80%, with no substantial toxicity.
Imaging results of fluorescent probe (5 mu M) in RAW264.7 cells under different conditions
To explore the biological applicability of the probe, we used the probe BODIPY-T to detect superoxide anion content at the cellular level. RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM). RAW264.7 cells were seeded in 4-well dishes (1.3X 10) prior to fluorescence imaging4One/well), cultured for 1 day.
The first group of RAW264.7 cells was incubated with BODIPY-T only.
A second set of RAW264.7 cells was treated with PMA for 30 min and then BODIPY-T (5. mu.M) in medium for 30 min.
A third group of RAW264.7 cells was treated with LPS for 30 min and then with BODIPY-T (5. mu.M) in medium for 30 min.
Finally all groups of cells were imaged by laser microscopy with an excitation wavelength of 488nm and a collection band of 515-580 nm. The first group of control cells fluoresced weakly (FIG. 12a) and the second group of cells pre-treated with PMA, observed intense fluorescence in the green channel (FIG. 12 b). In the third group of cells pretreated with LPS superoxide anion inducer, intense green fluorescence was also observed (FIG. 12 c). The results of the fourth group of LPS-treated cells followed by the Tiron-treated cells showed significantly weaker fluorescence intensity than the second and third groups (FIG. 12d), indicating that BODIPY-T could detect the difference in superoxide anion content in live cells. The results prove that BODIPY-T can be used as a good fluorescent probe to selectively detect superoxide anions in living cells.
Wherein:
a1, b1, c1 and d1 in FIG. 12 all represent bright field images in the cell;
a2, b2, c2 and d2 in FIG. 12 all represent images of fluorescence images in cells;
a3, b3, c3 and d3 in fig. 12 all represent the Merge map in the cell.
To facilitate a more intuitive viewing of fig. 6-12, the two-dimensional code of the color artwork of fig. 6-12 is appended. Specifically, the two-dimensional code of fig. 3 is scanned by the handheld terminal.
The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (10)
1. A BODIPY fluorescent probe for specifically recognizing superoxide anion is characterized in that: the molecular formula of the fluorescent probe is C33H30BF2N2O4P, the structural formula is as follows:
2. the method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
dissolving a mixture of T-Br, BODIPY-COOH and DBU in anhydrous tetrahydrofuran to prepare a mixed solution A, stirring at room temperature under the protection of argon, and performing reduced pressure spin drying on the solvent after the reaction is finished; further purifying by column chromatography, eluting with eluent to obtain probe BODIPY-T;
wherein the structural formula of T-Br is
Wherein the structural formula of BODIPY-COOH is shown as
Wherein DBU has a structural formula of
3. The method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 2, wherein the method comprises the following steps: the preparation method of the T-Br comprises the following steps:
s1, mixing and reacting raw materials: dissolving a mixture of T-OH, NBS and DMTU in dichloromethane to prepare a mixed solution B with the volume molar concentration of 0.1-1.0mol/L, and stirring at room temperature to obtain a reaction solution;
s2, preparation of intermediate T-Br: washing the reaction solution with water for 3 times, drying dichloromethane with anhydrous sodium sulfate, and finally, carrying out decompression spin-drying on the solvent to obtain an intermediate T-Br;
wherein the structural formula of T-OH is
Wherein NBS has the structural formula
Wherein the structural formula of DMTU is
4. The method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 3, wherein the method comprises the following steps: the eluent is a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 2: 1.
5. The method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 4, wherein the method comprises the following steps: the molar ratio of T-Br, BODIPY-COOH and DBU is 9:5: 10.
6. The method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 5, wherein the method comprises the following steps: the molar ratio of T-OH, NBS and DMTU was 1:1.5: 0.45.
7. The method for preparing BODIPY fluorescent probe capable of specifically recognizing superoxide anion according to claim 6, wherein the method comprises the following steps: the volume molar concentration of the mixed solution A is 0.5-1.0 mol/L.
8. Use of the BODIPY-based fluorescent probe specifically recognizing a superoxide anion of claim 1 in a detection reagent for detecting, recognizing, measuring the concentration of the superoxide anion in water or a marker in an environment or a biological sample.
9. The use of BODIPY-type fluorescent probes capable of specifically recognizing superoxide anion as claimed in claim 8, wherein the BODIPY-type fluorescent probes are characterized in that: the application of the superoxide anion in preparing detection reagents or markers of normal cells and living bodies.
10. The use of BODIPY-type fluorescent probes capable of specifically recognizing superoxide anion as claimed in claim 8, wherein the BODIPY-type fluorescent probes are characterized in that: the detection method for detecting the superoxide anion by the BODIPY fluorescent probe of the superoxide anion comprises the following steps:
measuring the fluorescence intensity of superoxide anion at the wavelength of 530nm by using a fluorescence spectrophotometry with 480nm as an excitation wavelength; the detection limit of the sample was 0.062 micromolar.
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